1. Field of the Invention
This invention is related to and has among it objects the provision of improved assay methods for detection of organisms. Primarily, the present invention is directed to assay methods involving immuno-reactions such as, for example, agglutination assays, enzyme immunoassays, and the like.
Agglutination assays have been used to detect the presence of an organism in a specimen, the assays finding extensive use in serological testing, typing of organisms, and bacterial culture confirmation. A variety of agglutination protocols have been used. In direct agglutination assays an antigen is present on the surface of a cell. Addition of an antibody specific for the antigen causes the cells to bind together through antigen antibody complexing thus producing a precipitate (agglutinate). The formation of an agglutinate indicates a positive test in the agglutination assay.
In a variation of the above method antigen is absorbed onto or linked to a cell. Addition of antibody causes the cells containing the absorbed antigen to agglutinate.
In reverse passive agglutination antibody is linked or absorbed onto the surface of a cell or latex particle. When antigen is present in the test medium, the cells or particles having antibody on their surface will agglutinate. Other variations of agglutination assays are known in the art.
Another assay involving an immuno-reaction is the enzyme-linked immunoadsorbent assay (ELISA), which has found considerable popularity for detecting and quantitating an organism. In the ELISA, an antigen or antibody is labeled with an enzyme. The labeled member is combined with the organism after which the enzyme-labeled antigen-antibody complex is separated from free enzyme-labeled antigen or antibody. The enzymatic activity in the bound or free fraction is quantitated by the enzyme-catalyzed conversion of a relatively nonchromatic or non-fluorescent substrate to a highly chromatic or fluorescent product.
For assays involving antigens, such as bacterial antigens of intact bacteria, a pretreatment step of the bacterial culture is often required. The pretreatment step is necessary to provide a homogeneous suspension of the test antigens in the test medium and to expose antigens of interest for access to the reaction. Without the application of a pretreatment step clumps and strands of organisms are present in the test medium giving rise to uninterpretable control reactions. The coagulation lattice tends to adhere to these clumps and strands, making differentiation between the test and the control reactions difficult.
Various pretreatment methods for organisms which are to be subjected to an assay are known. For example, organisms to be tested may be boiled for at least five minutes prior to the testing. The boiling procedure is, however, time consuming and laborious. The boiling step is not easily controlled and destroys some proteins (denaturation) indiscriminately which may reduce the density of certain epitopes or denature them so that they are no longer able to react with specific antibody. Enzymatic pretreatment steps are also known, with proteases and nucleases having been employed.
2. Description of the Prior Art
The bacterial cell is discussed in "Bacterial Morphology and Ultrastructure," Joklik et al (eds.). Zinsser Microbiology, pages 28-47, Appleton-Century-Crofts, New York, New York (1976). Various pretreatments for optimal performance of an agglutination test are disclosed in the following: Anand et al., J. Clinical Micro., 12:15-17 (1980); Lue et al, J. Clin. Micro. 8:326-328 (1978); Stockman et al, J. Clin. Micro., 16:965-967 (1982); Arko et al, J. Clin. Micro. 9:517-519 (1979). Lysis of gram negative organisms and the role of ethylenediaminetetraacetic acid in such lysis is discussed by Repaske, Biochimica et Biophysicia Acta, 30:225-232 (1958). Improved techniques for the preparation of bacterial lipopolysaccharides is discussed by Johnson et al, Can. J. Microbiol., 22:29-34 (1976).